Conformational changes involved in G-protein-coupled-receptor activation

Little is known about the nature of the conformational changes that convert G-protein-coupled receptors (GPCRs), which bind diffusible ligands, from their resting into their active states. To gain structural insight into this process, various laboratories have used disulfide cross-linking strategies involving cysteine-substituted mutant GPCRs. Several recent disulfide cross-linking studies using the M₃ muscarinic acetylcholine receptor as a model system have led to novel insights into the conformational changes associated with the activation of this prototypical class I GPCR. These structural changes are predicted to involve multiple receptor regions, primarily distinct segments of transmembrane helices III, VI and VII and helix 8. Given the high degree of structural homology found among most GPCRs, it is likely that these findings will be of considerable general relevance. A better understanding of the molecular mechanisms underlying GPCR activation might lead to novel strategies aimed at modulating GPCR function for therapeutic purposes.

Introduction

The superfamily of G-protein-coupled receptors (GPCRs) represents the largest group of cell-surface receptors found in nature [1]. The transmembrane (TM) core of these receptors, consisting of a bundle of seven TM helices (TMs I–VII), shows a very high degree of structural conservation, at least among class I GPCRs 2, 3, 4, 5, 6. Class I GPCRs represent by far the largest GPCR subfamily, containing ∼670 full-length human receptor proteins [1]. Members of this receptor family, including, for example, different dopamine, serotonin, adrenergic, muscarinic, prostanoid, cannabinoid, opioid and somatostatin receptor subtypes, are the target of nearly half of the clinically important drugs [1]. Such drugs are widely used in the treatment of psychiatric and cardiovascular disorders and many other pathophysiological conditions [1].

The conformational changes involved in the activation process of class I GPCRs are currently being studied by many laboratories. The most common biochemical and biophysical approaches that have been used to monitor such structural changes are listed in Table 1 (for a recent review, see Ref. [7]). The structural insights gained from these studies might lead to the design of novel classes of drugs that can modulate specific GPCR signaling pathways.

Biochemical and biophysical analysis of bovine rhodopsin, a class I GPCR that is unique in that its endogenous ligand (11-cis-retinal) is covalently bound to the receptor protein, has elucidated the light-induced changes in receptor conformation in considerable detail 8, 9, 10, 11, 12, 13. The vast majority of these studies were carried out with mutant versions of rhodopsin in the solution state.

A recent study [14] reported the crystal structure of a photoactivated intermediate of bovine rhodopsin at relatively low resolution (4.15 Å). The authors concluded that the resting and the activated states of bovine rhodopsin showed only minor structural differences [14]. By contrast, a great amount of biophysical and biochemical evidence indicates that rhodopsin activation is associated with more significant structural changes 8, 9, 10, 11, 12, 13. Possible explanations for the surprising observation that no large-scale structural changes were detected might be crystal-packing constraints [14] or the existence of multiple substates of metarhodopsin II endowed with different degrees of activity [15].

In contrast to the photoreceptor rhodopsin, much less is known about the agonist-induced conformational changes that occur in GPCRs activated by diffusible ligands. Using fluorescence-based biophysical studies carried out with purified, mutationally modified versions of the β₂-adrenoceptor, several activity-dependent changes at the intracellular receptor surface have been detected 16, 17.

More recently, we employed a disulfide cross-linking strategy that enables the identification of activity-dependent conformational changes using receptors present in their native membrane environment (in situ) 18, 19, 20, 21, 22, 23, 24. For these studies, we used the rat M₃ muscarinic acetylcholine (ACh) receptor, a prototypic class I biogenic amine GPCR that preferentially activates G_q-type G proteins, as a model system. Similar approaches have recently been developed to study agonist-induced conformational changes in the thyrotropin-releasing hormone receptor type I [25] and parathyroid hormone receptors [26]. Here, we provide an overview of the outcome of several recent disulfide cross-linking studies carried out with the M₃ receptor and provide a model of the structural changes associated with M₃-receptor activation. In addition, the conclusions drawn from these studies, including potential caveats associated with disulfide cross-linking approaches, are discussed in the context of the activation mechanism proposed for bovine rhodopsin, the prototypic class I GPCR.